Problem with stable overexpression cell lines - (Jul/28/2009 )

I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:

1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.

2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well

3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.

20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !

-jueduimeinv-

jueduimeinv on Jul 28 2009, 10:45 AM said:

Hi, All

I am encounting problems in generating stable cell line that overexpresses the gene XXX. The host cells are B16F10, vector I used is pSecTag2B from invitogen with CMV as promoter, selection marker is Zeocin. However, all the stable clones I selected under zeocin are not expressing my gene XXX. By troubleshooting, I exclude the following factors that may cause the failure:

1. the concentration of zeocin I used for selection of stable cell lines is OK because the same concentration of zeocin kills all wildtype B16F10 cells within 2 weeks.

2. the protein expression can be detected after transient transfection, which means that the plasmid is OK as well

3. the gene XXX has been overexpressed in other cell lines by other people although the vector they used is different, therfore my protein should not be very toxic to cells.

20 clones were picked up and cultured for few passages, BUT none of them give me positive signal by Western Blot. I am looking for your comments and advice. Thank you very much in advance !

I have encountered similar problems before. Depending on the number of genomic integrations, your expression may just be low. Cells generally don't need more than 1 copy of a resistance gene to survive selection, but one copy of your target gene may yield low expression. Check your cells by qRT-PCR to see if your transcript is being made and how much transcript is being made relative to other genes. Compare this to your transient transfection results as this can clue you in on the general expression level.
If the expression is there, but low, try repeating with increased zeocin concentration or a higher multiplicity of infection or higher plasmid concentration depending on how you are making your stables.

-Dr Teeth-

You might try a different promoter. I don't know much about cell line generation, but I know that in vivo, the CMV promoter is prone to silencing. A ubiquitin or EF1a promoter might be worth a shot.

-gfischer-

gfischer on Jul 28 2009, 10:04 AM said:

You might try a different promoter. I don't know much about cell line generation, but I know that in vivo, the CMV promoter is prone to silencing. A ubiquitin or EF1a promoter might be worth a shot.

could be the promoter, but if it were, then transient expression levels would probably be low as well.

copy number is important...stables will have far less compared to transient

some stables even when the plasmid integrates become genetically instable and are prone to silencing like previously mentioned.

some plasmid integrate close to silencer elements and so the gene doesn't express well if at all. inserting next to enhancer elements yields robust expression.

with stables, and similar to transgenic animals, there is always a possibility that the cell type you are using has a compensatory mechansim to regulate that particular proteins levels...your human gene under control of the CMV pro expresses above what the cell requires and so either the CMV promoter is silenced or the endogenous promoter for the gene in that cell type is silenced...on a western, it will look like no induction or overexpression is occuring. this can be somewhat validated if the mRNA is detected by using gene specific primers. does your antibody detect both endogenous protein and your protein or will it only detect your protein?

although excessive overexpression of your gene from the CMV looks nice and is convincing when showing the level of expression, excessive levels also bring artifacts to the cell's you will analyze...excess protein can cause accumulations in the ER and upregulate the UPR or take on some dominant negative effect.

1 and 2 fold overexpression should work well for most assays and limits artifacts of overrexpression